foods Article The Effect of Astaxanthin-Rich Microalgae “Haematococcus pluvialis” and Wholemeal Flours Incorporation in Improving the Physical and Functional Properties of Cookies A. K. M. Mofasser Hossain 1,2, Margaret A. Brennan 1, Susan L. Mason 1, Xinbo Guo 3, Xin An Zeng 3 and Charles S. Brennan 1,2,* ID 1 Centre for Food Research and Innovation, Department of Wine, Food and Molecular Biosciences, Lincoln University, Lincoln 7647, New Zealand; [email protected] (A.K.M.M.H.); [email protected] (M.A.B.); [email protected] (S.L.M.) 2 Riddet Institute, Palmerston North 4442, New Zealand 3 School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China; [email protected] (X.G.); [email protected] (X.A.Z.) * Correspondence: [email protected]; Tel.: +64-3-4230-637 Received: 23 May 2017; Accepted: 21 July 2017; Published: 26 July 2017 Abstract: Marine-based food supplements can improve human nutrition. In an effort to modulate glycaemic response and enhance nutritional aspects, marine-derived algal food rich in astaxanthin was used in the formulation of a model food (wholemeal cookie). Astaxanthin substitution of cookies made from three flours (wheat, barley and oat) demonstrated a significant reduction in the rate of glucose released during in vitro digestion together with an increase in the total phenolic content (TPC) and antioxidant capacity of the food. The significantly (p < 0.005) lower free glucose release was observed from cookies with 15% astaxanthin, followed by 10% and then 5% astaxanthin in comparison with control cookies of each flour. Total phenolic content, DPPH radical scavenging and Oxygen Radical Absorbance Capacity (ORAC) value also notably increased with increase in astaxanthin content. The results evidence the potential use of microalgae to enhance the bioactive compounds and lower the glycaemic response of wholemeal flour cookie. Keywords: microalgae; Hematococcus pluvialis; astaxanthin; bakery products; glycaemic response; antioxidant 1. Introduction Whole-grains such as wheat, barley and oat make a substantial contribution to our diet. They contain a significant amount of bioactive compounds such as fibre, minerals, vitamins and phytochemicals [1,2] and as such mayplay a major role in enhancing human health by reducing the risk of diabetes [3,4] and cancer [5], while also regulating serum cholesterol [6] and stimulating beneficial gut microbiota [7]. In recent years there has been an increased interest in the utilisation of whole-grain food materials as well as fibre rich ingredients, in cereal products, including bread [8], extruded snack products [9,10], and pasta [11,12]. These pieces of research have investigated the impact of wholegrains and fibre on both the physicochemical characteristics of cereal food products as well as their nutritional quality. A recent review on this subject illustrated that the incorporation of fibre rich ingredients into cereal products often results in negative consumer acceptability [13]. There therefore remains a challenge to both utilise wholegrain cereal products as well as functional food ingredients such a fibre rich materials, into mainstay food products. Foods 2017, 6, 57; doi:10.3390/foods6080057 www.mdpi.com/journal/foods Foods 2017, 6, 57 2 of 10 Recent research into functional food ingredients has shown an interest in the development of foods containing seaweed or algal materials [14,15]. These materials have been part of the human diet since 600 BC [16] and they have a role of diet in sustaining human due to their diverse range of nutrients and bioactive compounds; such as polysaccharides, proteins, polyunsaturated fatty acids, minerals and significant amounts of antioxidants [17,18]. One such material is Haematococcus pluvialis, a single-cell microalgal strain, which is rich source of astaxanthin (10,000–40,000 mg/kg) and associated bioactive ingredients including dietary fibre [19]. Several cell culture and animal studies have reported that astaxanthin has potent antioxidant activity 10 times higher than other carotenoids such as β-carotene, lutein, and zeaxanthin, and 500 times higher than vitamin E [20–22]. Carotenoids play a role in preventing or delaying degenerative diseases such as cancer and atherosclerosis diseases [23–25], and may be useful in the development of functional foods [15]. There is a paucity of information regarding combining the nutritional compounds of marine-based material and whole-grains. Therefore, the present study is the first to show the glycaemic glucose equivalents (GGE) as a predictor of glycaemic response, antioxidant capacities and physical properties of cereal and Hematococcus pluvialis in a model food. 2. Materials and Methods 2.1. Sample Collection and Preparation Driedmicroalgae Hematococcus pluvialis was provided by Supreme Biotechnologies Ltd. (Nelson, New Zealand) and ground using a grinder (AutoGrinder, M-EM0415, Sunbeam Corp Ltd., Auckland, New Zealand). The ground material was sieved through a 0.5 mm screen to obtain flour. Wholemeal wheat (Champion Flour, Auckland, New Zealand), barley (Ceres Organics, Auckland, New Zealand) and oat flours (Ceres Organics, Auckland, New Zealand) were purchased locally. 2.2. Cookie Preparation Cookies were prepared following the standard American Association of Cereal Chemistry (AACC) method 10–50D [26] with slight modification. Table1 illustrates the dry ingredients used (sugar, salt and sodium bicarbonate). All dry ingredients (except flour) were mixed in an electric mixer (Breville, Melbourne, Australia) with vegetable shortening (Kremelta, Peerless foods, Braybook, Australia) for 3 min on speed 1. Dextrose solution (8.9 g dextrose anhydrous in 150 mL water) and distilled water were added to the mixer and mixed for a further 1 min on speed 2 with scraping down every 30 s. The flour was added and mixed for 2 min with scraping down every 30 s. The experimental samples were prepared by replacing the wholemeal flour with astaxanthin powder 5%, 10% and 15%. The cookie dough was rolled to a 6 mm thickness using measuring roller and cut with a 57 mm diameter cookie cutter. The cookies were placed on metal trays and baked in a preheated electric oven (BAKBAR turbofan convection oven, E3111, Moffat Pty Ltd., Rolleston, New Zealand) for 8 min at 180 ◦C. The cookies were cooled at room temperature, placed in air-tight plastic bags and stored at room temperature for 24 h prior to laboratory analysis. Table 1. Model food formulation. Wholemeal Astaxanthin Sample Other Ingredients Flour (g) Powder (g) Vegetable shortening (64.0 g), sugar (130 g), Control 225.00 - salt (2.1 g), sodium bicarbonate (2.5 g), dextrose solution (33 g), water (16 g) 5% Astaxanthin powder 213.75 11.25 Vegetable shortening (64.0 g), sugar (130 g), 10% Astaxanthin powder 202.50 22.50 salt (2.1 g), sodium bicarbonate (2.5 g), 15% Astaxanthin powder 191.25 33.75 dextrose solution (33 g), water (16 g) Foods 2017, 6, 57 3 of 10 2.3. Physical Characteristics Cookie diameter (mm) and thickness (mm) were measured using calipers (INSIZE digital caliper, series 1112, INSIZE Inc., Loganville, GA, USA). The colour of the cookie samples were measured in terms of Comission Internationale de l’Eclairage (CIE) L*, a* and b* systems by using a colorimeter (Konica Minolta, Chroma Meters CR-210, Tokyo, Japan). The colour differences of the cookies were calculated by the following equation. q DE = (DL∗)2 + (Da∗)2 + (Db∗)2 2.4. Texture The hardness of the cookies (fracture force) was measured by using a texture analyser (TA.XT plus Texture Analyser, Stable Micro Systems, Godalming, UK) with a 3-point bend rig. The analyser was set at a load cell 50 kg; pre-test speed 2 mm/s; test speed 5 mm/s; post-test speed 10 mm/s; return to start mode. The whole cookies were placed on the support ring and the probe moved downward until the samples were broken. The peak force (kg) was recorded as hardness. Measurements were made in triplicate. 2.5. Moisture Moisture content of the cookie samples were measured after drying cookie ground samples (2 g) overnight in an oven at 105 ◦C. 2.6. Determination of Total Phenolic Content The content of total phenolics of samples was measured by Folin-Ciocalteu reagent (mixture of phosphotungstic and phosphomolybdic acid; that is reduced by phenolics forming a blue complex) using the method described by Floegel et al., 2011 [27] with some modifications. The ground samples (1 g) were dispersed in 20 mL of 70% methanol (by placing on a stirrer overnight). The sample mixture was centrifuged at 700 g Relative centrifugal force (RCF) for 10 min and the supernatant collected to determine the total phenolics. Crude extracts (0.5 mL) were mixed thoroughly with freshly prepared 0.2 N Folin-Ciocalteu’s reagent (2.5 mL), followed by 2.0 mL of 7.5% sodium carbonate (Na2CO3) and incubated in the dark for 2 hours. The absorbance reaction mixture was measured at 760 nm. Gallic acid (gallic acid, 97%, CAS: 149-91-7, Sigma-Aldrich, St. Louise, MO, USA) was used as a standard and results were expressed as mg gallic acid equivalent (GAE) per g sample. 2.7. Antioxidant Properties The antioxidant capacity of the samples was measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as described by Floegel et al., 2011 [27] with some modifications. Briefly, 0.5 mL of crude extract was mixed with freshly prepared 1 mL of 0.1 mM methanolic DPPH (CAS: 1898-66-4, Sigma-Aldrich, St. Louise, MO, USA) solution and incubated in the dark at room temperature for 30 min. The reaction mixture absorbance was measured at 517 nm. In order to calculate the DPPH radical scavenging capacity, trolox (CAS: 53188-07-1, ACROS Organics™, Morris, NJ, USA) was used as a standard and result were expressed as µmol trolox equivalent (TE) per g sample.